Synthetic lipid mixtures for the preparation of a reconstituted surfactant

ABSTRACT

The invention relates to reconstituted surfactants consisting of artificial phospholipids and peptides able to lower the air-liquid surface tension, more particularly to reconstituted surfactants, comprising special phospholipid mixtures and artificial peptides which are analogues of the natural surfactant SP-C protein for the treatment of respiratory distress syndrome (RDS) and other diseases relates to pulmonary surfactant dysfunctions.

The present invention relates to reconstituted surfactants consisting ofphospholipids and artificial peptides able to lower surface tension atthe air-liquid interface. In particular, the invention relates toreconstituted surfactants comprising particular phospholipid mixturesand artificial peptide analogues of the natural surfactant SP-C proteinfor the treatment of respiratory distress syndrome (RDS) and otherdiseases related to pulmonary surfactant dysfunctions.

BACKGROUND OF THE INVENTION

Pulmonary surfactant lowers the surface tension arising at theair-liquid interface of the internal alveolar wall, thus preventing thelungs from collapsing at the end of expiration. Surfactant deficiency isa dysfunction which commonly affects preterm infants and causes RDS, adisease which can be effectively treated with natural surfactantsextracted from animal lungs. The main constituents of these surfactantpreparations are phospholipids, such as1,2-dipalmitoyl-sn-glycero-3-phosphocholine commonly known asdi-palmitoyl-phosphatidyl-choline (DPPC), phosphatidylglycerol (PG) andsurfactant hydrophobic proteins B and C (SP-B and SP-C). The hydrophilicsurfactant proteins SP-A and SP-D, which are C-type (Ca²⁺-dependent)collagenous lectins and thought to act primarily in the host-defencesystem, are normally not included in the surfactant preparations due tothe organic solvent extraction procedures employed. Modified naturalsurfactant preparations obtained from animal tissues are used in currenttherapeutical practice. These preparations usually consist of theaforementioned components with the exception of hydrophilic proteins,which are removed upon extraction with organic solvents.

Owing to the drawbacks of the surfactant preparations from animaltissues, such as the complexity of the manufacturing and sterilizationprocesses and possible induction of immune reactions, attempts toprepare artificial surfactants have been made.

In the strict sense of the word, artificial surfactants are mixtures ofphospholipids only or mixtures of phospholipids and other syntheticlipids. Reconstituted surfactants are artificial surfactants added withhydrophobic proteins—either isolated from animal tissues or obtainedthrough recombinant techniques—or synthetic peptidic derivatives of suchproteins.

The properties and the activity of reconstituted surfactants greatlydepend not only on the protein/peptide components, but also on thecomposition of the phospholipid mixture.

DESCRIPTION OF THE INVENTION

It has now been found that dipalmitoyl phosphatidylcholine (DPPC) inadmixture with specific palmitoyl oleyl phospholipids is an idealvehicle for the artificial peptides commonly used in reconstitutedsurfactants as analogues of natural surfactant proteins SP-C and/orSP-B. In particular, reconstituted surfactants comprising the SP-Canalogues disclosed in WO 00/47623 in combination with DPPC and apalmitoyl oleyl phospholipid—preferably selected from palmitoyl oleylphosphatidylglycerol (POPG) or a mixture of POPG with palmitoyl oleylphosphatidylcholine (POPC)—in weight ratios ranging from 80:20 to 60:40have been found to lower the surface tension and the viscosity of thepreparations obtained therefrom. Contrary to what is reported in theliterature, it has also surprisingly been found that the addition ofpalmitic acid (PA) is useless and, what's more, in some cases can lowerthe in vivo surfactant's activity.

Accordingly, the present invention relates to reconstituted surfactantscomprising mixtures essentially consisting of dipalmitoylphosphatidylcholine (DPPC) and palmitoyl oleyl phosphatidylglycerol(POPG) or a mixture thereof with palmitoyl oleyl phosphatidylcholine(POPC) in weight ratios ranging from 80:20 to 60:40 and artificialpeptide analogues of natural surfactant proteins SP-C and/or SP-B. Thesurfactant of the invention is devoid of palmitic acid.

The weight ratio between DPPC and POPG ranges preferably from 75:25 to65:35, and is more preferably 68:31. In the case of DPPC:POPG:POPCmixtures, the phospholipids are preferably used in weight ratios of60:20:20 or 68:15:16.

Any artificial peptide analogue of natural surfactant proteins SP-Cand/or SP-B can be advantageously used, such as those disclosed in WO89/06657, WO 92/22315 and WO 95/32992. Preferred is the use of SP-Canalogues having the following general formula (I), the amino acidsbeing represented with the one-letter code,F_(e)G_(f)IPZZPVHLKR(X_(a)B)(X_(b)B)_(n)(X_(c)B)_(m)X_(d)GALLΩGL   (I)wherein:

-   -   X is an amino acid selected from the group consisting of I, L,        nL (norleucine);    -   B is an amino acid selected from the group consisting of K, W,        F, Y, ornithine;    -   Z is S optionally substituted with acyl groups containing 12-22        carbon atoms linked to the side chain via an ester or thio-ester        bond, respectively;    -   Ω is an amino acid selected from the group consisting of M, I,        L, nL;    -   a is an integer from 1 to 19;    -   b is an integer from 1 to 19;    -   c is an integer from 1 to 21;    -   d is an integer from 0 to 20;    -   e is 0 or 1;    -   f is 0 or 1;    -   n is 0 or 1;    -   m is 0 or 1;        with the following provisos:    -   n+m>0;    -   f≧e,    -   (X_(a)B)_(n)(X_(b)B)_(n)(X_(c)B)X_(d) is a sequence having a        maximum of 22 amino acids, preferably from 10 to 22.

Even more preferred is the use of peptides of formula (II)IPSSPVHLKRLKLLLLLLLLILLLILGALLΩGL   (II)wherein:

-   -   Ω is an amino acid selected from the group consisting of M, I,        L, nL and wherein serine can optionally be acylated, for example        with palmitoyl.

The hereinafter reported peptide (SP-C33), in the non-acylated form, isthe most preferred of the invention,IPSSPVHLKRLKLLLLLLLLILLLILGALLMGL (SP-C33)

Peptides of formula (I) may be prepared by means of conventional peptidesynthesis or recombinant techniques as disclosed in WO 00/47623.

Acylated peptides are preferably synthesized by reacting the peptideswith an acyl chloride in pure trifluoroacetic acid for 10 hours at roomtemperature, followed by quenching with 80% aqueous ethanol.

The activity of the reconstituted surfactants of the invention inreducing surface tension has been evaluated both in vitro and in vivo.In particular, the in vivo results clearly show that the reconstitutedsurfactants of the present invention are able to increase tidalvolume—which is in turn an index of the pulmonary expansion capacity—ina significantly higher extent than the surfactants obtained with themixtures of DPPC, PG and PA commonly used in the prior art.

The reconstituted surfactants of the invention may comprise SP-B orpolymixins, in particular polimixin B, as SP-B analogues.

The surfactant can be prepared by mixing solutions or suspensions ofpeptides and phospholipids and by subsequently drying the mixture. Ifnecessary, the dried mixture can be re-suspended, dispersed oradministrated as such to subjects which require a treatment forsurfactant deficiency.

In the case of aerosol administration, it will be necessary to combinesmall surfactant particles with a suitable inert propellant. Otheradministration forms, such as vapourisation or nebulization of stablesurfactant solutions/suspensions, are also within the scope of thepresent invention.

The following examples illustrate the invention in greater detail.

EXAMPLES

Experimental Section

Materials

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), eggphosphatidylglycerol (PG), palmitic acid (PA), and polymyxin B (P×B)sulfate were utilized to prepare the following lipid mixtures and weightratios: DPPC/PG/PA, 68:22:9; DPPC/POPG/PA, 68:22:9; DPPC/POPC/POPG,60:20:20; DPPC/POPG/POPC/PA, 57:19:19:5; DPPC/POPC/POPG, 68:16:15 andDPPC/POPG, 68:31.

SP-C33 was synthesized and purified as described in WO 00/47623.

Preparation of Synthetic Surfactants

SP-C33 and lipids in peptide/lipid weight ratios of 0.02:1 were mixed inchloroform/methanol 98:2 (v/v), the solvents were evaporated and theresulting peptide/lipid films were subsequently hydrated in 150 mM NaClby repeated sonication, at a lipid concentration of 80 or 35 mg/ml. Thesurfactant samples used for analyses in the pulsating bubblesurfactometer were diluted in saline to working concentrations (10 mg/mlsurfactant). In some experiments P×B was added up to a finalconcentration of 2% (w/w) of the lipid concentration. The surfactantwhich contained SP-C33 in DPPC/PG/PA (68:22:9) provided very viscoussuspensions at 80 mg/ml and was difficult to administer to theexperimental animals.

Two well known modified natural surfactants, Curosurf (ChiesiFarmaceutici) and Survanta (Abbott) were administered according to themanufacturer's instructions. Curosurf was suspended at 80 mg/ml and 2.5ml/kg body weight was administered; Survanta was suspended at 25 mg/mland 4 ml/kg body weight was administered.

Pulsating Bubble Experiments

The dynamic surface properties of SP-C33 surfactants with and without 2%(w/w) P×B were evaluated by a pulsating bubble surfactometer. For theseexperiments, the surfactant was suspended in saline at a concentrationof 10 mg/ml and analysed at 37° C. The sensitivity towards inhibitionwas tested by adding 40 mg/ml albumin to the surfactant suspension. Abubble communicating with ambient-air was created in a plastic testchamber containing approximately 20 μl of the sample fluid. The bubbleradius was oscillated at a rate of 40 cycles/min. from a maximum of 0.55to a minimum of 0.40 mm, corresponding to a 50% cyclic surfacecompression. Surface tension values at minimum and maximum bubble size(γ_(min), γ_(max)) were recorded over 5 min. pulsations.

In Vivo Experiments

The surfactant mixtures were assayed in premature newborn rabbits,obtained by hysterectomy at the gestational age of 27 days (term: 31days). The animals were tracheotomized at birth, kept in plethysmographyboxes at 37° C. and ventilated in parallel with 100% oxygen at afrequency of 40 breaths/min. and a 50% inspiration time. Treated animalsreceived 2.5 ml/kg or 4 ml/kg of the above surfactant preparationthrough a tracheal cannula. Litteimates which did not receive anysurfactant preparation were used as controls. After instilling thesurfactant, peak pressure was first raised to 35 cmH₂O per 1 min., tofacilitate the distribution of the surfactant in the lungs, then loweredto 25 cmH₂O. The animals were then ventilated with a peak pressure of 25cmH₂O for 15 min., thereafter the pressure was lowered first to 20 cmH₂Ofor 5 min., then to 15 cmH₂O for 5 min. and raised again to 25 cmH₂O for5 min. Tidal volumes were measured at 5 min. intervals with apneumotachograph connected to each plethysmograph box.

At the end of the established ventilation period, the animals weresacrificed by intracerebral lidocain injection. Their abdomen was openedand the diaphragm position was inspected for pneumotorax evidences.

Example 1 SP-C33 In Vitro Superficial Activity in Different LipidMixtures

The dynamic surface properties of 2% (w/w) SP-C33 in DPPC/PG/PA(68:22:9), DPPC/POPC/POPG (60:20:20), DPPC/POPC/POPG (68:16:15) andDPPC/POPG (68:31) were evaluated with a pulsating bubble surfactometer,which showed γ_(min)<2 mN/m after 5 min. pulsation for all the mixturesand γ_(max)<40 mN/m for all the mixtures except for SP-C33 in DPPC/PG/PA(68:22:9), whose γ_(max) was 48 mN/m.

Example 2 In Vivo Optimal Effect Without PA

To evaluate the relevance of the lipid composition, we compared the invivo effects of SP-C33 in the mixtures DPPC/PG/PA (68:22:9),DPPC/POPG/PA (68:22:9) and DPPC/POPG (68:31). SP-C33 in DPPC/POPG(68:31) showed a higher effect than the other two mixtures. The datashowed a marked increase in tidal volumes after treatment with SP-C33 inDPPC/POPG (68:31). They also showed that, if the DPPC and acid lipidcontent is constant, the presence of PA reduces the effect of thetreatment. A negative effect of PA on the in vivo activity of theSP-C33-based surfactant is further confirmed by the effect (V_(T)=12 a15 min. e V_(T)=14 a 25 min.) of SP-C33 in DPPC/POPC/POPG/PA(57:19:19:5) which is slightly lower than that of SP-C33 inDPPC/POPC/POPG (60:20:20).

1-11. (canceled)
 12. A reconstituted surfactant consisting of anartificial peptide and phospholipids wherein the artificial peptide is aSP-C analogue of formula (I)F_(e)G_(f)IPZZPVHLKR(X_(a)B)(X_(b)B)_(n)(X_(c)B)_(m)X_(d)GALLΩGL   (I)wherein: X is an amino acid selected from the group consisting of I, L,and nL (norleucine); B is an amino acid selected from the groupconsisting of K, W, F, Y, and ornithine; Z is S optionally substitutedwith acyl groups comprising 12-22 carbon atoms linked to the side chainvia an ester bond; a is an amino acid selected from the group consistingof M, I, L, and nL; a is an integer from 1 to 19; b is an integer from 1to 19; c is an integer from 1 to 21; d is an integer from 0 to 20; e is0 or 1; f is 0 or 1; n is 0 or 1; m is 0 or 1; n+m>0; f≧e; and(X_(a)B)(X_(b)B)_(n)(X_(c)B)_(m)X_(d) is a sequence having a maximum of22 amino acids; in combination with dipalmitoyl phosphatidylcholine(DPPC) and a palmitoyl oleyl phospholipid selected from palmitoyl oleylphosphatidylglycerol (POPG) or a mixture thereof with palmitoyl oleylphosphatidylcholine (POPC) in weight ratios ranging from 80:20 to 60:40.13. The reconstituted surfactant according to claim 12 wherein theartificial peptide is a SP-C analogue of formula (II)IPSSPVHLKRLKLLLLLLLLILLLILGALLΩGL   (II) wherein Ω is a residue selectedfrom the group consisting of M, I, and nL.
 14. The reconstitutedsurfactant according to claim 13 wherein the artificial peptide isIPSSPVHLKRLKLLLLLLLLILLLILGALLMGL (SP-C33).
 15. The reconstitutedsurfactant according to claim 12 wherein the artificial peptide is incombination with DPPC and POPG in weight ratios ranging from 75:25 to65:35.
 16. The reconstituted surfactant according to claim 15 whereinthe artificial peptide is in combination with DPPC and POPG in a weightratio of 68:31.
 17. The reconstituted surfactant according to claim 12wherein the artificial peptide is in combination with DPPC, POPG andPOPC in a weight ratio of 60:20:20 to 68:15:16.
 18. A process forpreparing said reconstituted surfactant of claim 12 comprising: i)mixing a solution or a suspension of the peptide and the phospholipids;and ii) drying the mixture.
 19. A dispersion, a suspension or a drypowder comprising the reconstituted surfactant according to claim 12.20. A method for the treatment of one or more diseases related to apulmonary surfactant dysfunction, said method comprising administeringsaid reconstituted surfactant according to claim 12 to a patient in needthereof.
 21. The method according to claim 20 wherein the disease isrespiratory distress syndrome (RDS).